Isolation and characterization of 32P-labeled mitochondrial and cytosol ribosomal RNA from germinating wheat embryos

With the aim of preparing highly labeled material, the incorporation of [32P]-orthophosphate into mitochondral and cytosol ribosomal RNA was examined in germinating wheat embryos. Nucleic acids were extracted from mitochondria and from post-mitochondrial supernatant (cytosol) prepared from homogenates of viable embryos (8g) imbibed for 24h in a medium containing [32P]orthophosphate (100mCi). High-molecular-weight ribosomal RNA was selectively precipitated in the presence of 3M NaCl and was further resolved on sucrose density gradients and polyacrylamide gels. Both the mitochondrial and cytosol NaCl-insoluble RNA fractions were found to contain two major radioactive components, corresponding to the large (26S) and small (18S)rRNA species. On non-denaturing gels, these species had apparent molecular weights of 1.3 and 0.67 million daltons (cytosol) and 1.3 and 0.75 million daltons (mitochondrial). The individual, purified [32P]rRNA components (isolated from sucrose gradients) had specific activities of 2--3-10(6) cpm/A260 unit, and were suitable for analysis of nucleotide composition and sequence. By hydrolyzing the individual [32P]rRNA specimens with purified snake venom phosphodiesterase and resolving the products by two-dimensional paper chromatography, it was possible to determine the specific activities (cpm/micronmol) of the four major 5'-nucleotide constituents. The results indicated that there had been no differential 32P-labeling of the nuclear and mitochondrial pools of ribonucleoside 5'-triphosphates (rNTP) during the 24 h imbibition period; however, as previously observed in this system (Lau, R.Y., Kennedy, T.D. and Lane, B.G. (1974) Can. J. Biochem. 52, 1110--1123), there had been unequal 32P-labeling of the individual rNTPs in both the mitochondria and nucleus. The relative specific activities of the 5'-nucleotide constituents of the mitochondrial and cytosol rRNA species were essentially the same, and in the order pA congruent to pU greater than pG greater than pC. By making suitable corrections for these differences in specific activity, the nucleotide composition of each of the [32P]rRNA specimens could be calculated...